WO2019216465A1 - Composition for prevention of breast cancer comprising cyclic dipeptide and methods of preparation thereof - Google Patents

Abstract

The present invention relates to a composition for preventing breast cancer comprising conditioned media which is removed Lactobacillus used for fermentation and methods of preparation thereof. The cyclic dipeptide according to the present invention is separated from Lactobacillus sp. isolated from Korean Kimchi. The composition comprising cyclic dipeptide could be a conditioned media wherein cultured Lactobacillus cell is removed. The Lactobacillus cell removed conditioned media has prevention effect to breast cancer.

Description

COMPOSITION FOR PREVENTION OF BREAST CANCER COMPRISING CYCLIC DIPEPTIDE AND METHODS OF PREPARATION THEREOF

The present invention related to Composition for prevention of breast cancer comprising cyclic dipeptide and methods of preparation thereof

Lactic acid bacteria (LAB) are known as Gram-positive bacteria that produce several types of organic acids as the predominant metabolites through metabolizing sugars. LAB are widely utilized as the prominent probiotics since they are benefit for host animals by improving its intestinal microbial balance ^[1]. Additionally, this group of probiotic bacteria have been highly selected strains, including Lactobacillus spp., Bifidobacterium spp., Streptococcus spp. et al., which are capable of surviving in animal stomach ^[2] , defining gut survival properties and can be ingested in fermented milk products or as a supplement ^[3]. Moreover, the significance of probiotic spectra of these kinds of strains have been well established ^[1,4,5]. Beside their beneficial effects for the nutrition and bowel activity, various disease prevention function of these probiotic strains were illustrated, especially related to specific diseases, such as diarrhea ^[6], lactose intolerance symptoms ^[7], constipation ^[8], inflammatory bowel disease ^[9,10] as well as cancers ^[3,11,12].

Most cancer prevention effect of LAB studies has been focused on colorectal cancer. It is generally accepted that LAB strains may promote several anticancer mechanism from relieving environmental carcinogenesis stimuli by altering the metabolic traits of intestinal microflora, degrading potential carcinogens ^[3] or regulating host's immune response ^[13]. Studies in subjects with probiotic LAB administration suggest that this might induce apoptosis in carcinogenesis pathways such as nuclear factor-kappa B signaling ^[14] or autophagic cell death ^[15]. Combination of different LAB strains were revealed to enhance 5-fluorouracil-induced apoptosis in colorectal cancer cell line ^[16]. LAB also displayed their cancer prevention capability by its antioxidant activities and epigenetic modification potential ^[17,18] especially by secreting short-chain-fatty acid, such as butyrate ^[19,20]. Interestingly, not only the heat inactive LAB show the anticancer property ^[21], but also the cell-free supernatants from LAB cultures are capable to inhibit colon cancer cell invasion by regulating matrix metalloproteinase-9 activity and zona occudens-1 ^[22].

Among those probiotic LAB mentioned above, Lb. plantarum has been regarded as the major strain that can be safely used in wide range of food production and preservation. Despite in fermented vegetable dairy or meat, Lb. plantarum also could be found in human gastrointestinal tract ^[23]. Lb. plantarum LBP-K10 was elucidated to be the most potent antimicrobial LAB, which exhibited the activity against multidrug-resistant bacteria, plant or human pathogenic fungi and influenza A virus, isolated from Korean traditional fermented vegetables, kimchi ^[24]. In the late stage of kimchi fermentation, a large amount of organic acid, which produced by the rapid increasing of Lb. plantarum LBP-K10, acidifies kimchi approximately pH ranging from 3.8 to 4.3 as the main acid substance. This aerotolerant bacteria can respire oxygen, but the consumed oxygen ultimately ends up as hydrogen peroxide due to the absence of respiratory chain or cytochromes. The peroxide can be help for excluding competing bacteria from the food source. Addition to this, anti-microorganism compound produced by this strain were continuously being characterized ^[25,26,27]. Moreover, the current studies explored the potential therapeutic effect of Lb. plantarum on various diseases, including recurrent Clostridium difficile-associated diarrhea ^[28], symptoms of irritable bowel syndrome ^[29] or even cardiovascular disease ^[30]. The anticancer activities of Lb. plantarum were also intensively investigated ^[31,32,33] in previous reports that their dead body or extractions of their metabolites also exert anticancer function ^[34,35].

Furthermore, a group of small molecules were fractionated and identified from a isolated Lb. plantarum LBP-K10 bearing significant anti-microorganism activities. These small molecules were observed to be proline-based cyclic dipeptides (CDPs) consisting of several kinds of diketopiperazines, including 2,3.-, 2,5 and 2,6-isomers ^[36,37]. With our previous reports, the existence of CDPs has been widely confirmed in other LAB species ^[38,39]. Besides the anti-microorganism activity, the tumor prevention effects of CDPs are also ascribed using a mouse model in this study. Additionally, CDPs were found to be capable of inducing cancer cell apoptosis ^[40,41] or preventing gene mutation ^[42].

However, most of anticancer studies have been mainly focused on gastrointestinal cancer cells, primarily on colon cancer and executed in vitro cell lines. Beacuse the anticancer effect of CDPs was well demonstrated and studies confirmed the rapid absorption of CDPs in animal ^[43] , it promptly us to investigate of the cancer prevention effect of the specific LAB stain, Lb. plantarum LBP-K10, on non-gastrointestinal cancer model. In this study, we discovered that oral administration heat inactive Lb. plantarum LBP-K10 conditioned medium (LBP-K10 CM) might effectively prohibit the progression of breast cancer in mouse transplantation model, possibly through apoptosis pathways induced by CDPs

The object of the present invention is to provide a composition of cyclic dipeptide for preventing breast cancer and methods of preparation thereof.

For the purpose of above object, the first aspect of present invention provides a composition for prevention of breast cancer comprising cyclic dipeptide. The said cyclic dipeptide could be produced from Lactobacillus sp. and be single or complex composition. Preferably, the said cyclic dipeptide is cyclo(Phe-Pro). The said Lactobacillus is, but not limited to, Lb. plantarum LBP-K10.

The said Lactobacillus sp. could be sterilized after production of cyclic dipeptide in a culture medium and removed from the culture medium. The said removal of Lactobacillus could be, but not limited to, by centrifugation.

The second aspect of present invention provides a methods of preparation of composition for prevention of breast cancer comprising the step of;

culturing Lactobacillus sp.in a culture medium;

sterilizing the Lactobacillus by heat treatment; and

removing the sterilized Lactobacillus sp. from the culture medium.

The said Lactobacillus is, but not limited to, preferably Lb. plantarum LBP-K10. The methods of preparation of composition for preventing breast cancer further comprise the step of concentrating the cyclic dipeptide.

The total amount of the active ingredient (cyclic dipeptide) to be administered preferably via the oral route using the pharmaceutical composition of the present invention will generally range from about 0.1 mg/kg to about 50 mg/kg body weight per day. Based upon standard laboratory techniques known to evaluate compounds useful for the treatment of hyper-proliferative disorders, by standard toxicity tests and by standard pharmacological assays for the determination of treatment of the conditions identified above in mammals, and by comparison of these results with the results of known medicaments that are used to treat these conditions, the effective dosage of the pharmaceutical compositions of this invention can readily be determined by those skilled in the art. The amount of the administered active ingredient can vary widely according to such considerations as the particular compound and dosage unit employed, the mode and time of administration, the period of treatment, the age, sex, and general condition of the patient treated, the nature and extent of the condition treated, the rate of drug metabolism and excretion, the potential drug combinations and drug-drug interactions, and the like.

Preference is given to an amount of the compound of cyclic dipeptide in the pharmaceutical composition from 20 to 2000 mg, preferably from 40 to 800 mg, more preferably from 50 to 600 mg.

The pharmaceutical composition according to the invention is administered one or more, preferably up to three, more preferably up to two times per day. Preference is given to an administration via the oral route.

Nevertheless, it may in some cases be advantageous to deviate from the amounts specified, depending on body weight, individual behavior toward the active ingredient, type of preparation and time or interval over which the administration is effect. For instance, less than the aforementioned minimum amounts may be sufficient in some cases, while the upper limit specified has to be exceeded in other cases. In the case of administration of relatively large amounts, it may be advisable to divide these into several individual doses over the day.

This pharmaceutical composition will be utilized to achieve the desired pharmacological effect by preferably oral administration to a patient in need thereof, and will have advantageous properties in terms of drug release, bioavailability, and/or compliance in mammals. A patient, for the purpose of this invention, is a mammal, including a human, in need of treatment for the particular condition or disease.

The pharmaceutical composition comprises suitable administration forms which deliver the compound of the invention in a rapid manner, for example tablets (uncoated or coated tablets), tablets which disintegrate rapidly in the oral cavity or capsules optionally filled with granules (for example hard or soft gelatin capsules), sugar-coated tablets, powders, sachets, granules, pellets, chewable tablets, dispersible tables, troches and lozenges.

Preference is given to tablets, granules, capsules optionally filled with granules, pellets, chewable tablets, dispersible tables, troches and lozenges. More preferably the application forms are tablets, granules and capsules optionally filled with granules. Most preferably the application form is a tablet.

A pharmaceutically acceptable excipient is any excipient which is relatively non-toxic and innocuous to a patient at concentrations consistent with effective activity of the active ingredient so that any side effects ascribable to the excipient do not vitiate the beneficial effects of the active ingredient.

Pharmaceutically acceptable excipients according to the invention are for example disintegrants, binders, lubricants, fillers, plasticizers, surfactants and wetting agents, film-forming agents and coating materials, and coloring agents for example pigments.

Disintegrants include, but are not limited to croscarmellose sodium, crospovidone, alginic acid, carboxymethylcellulose calcium, carboxymethylcellulose sodium, microcrystalline cellulose, hydroxypropyl cellulose, low substituted hydroxypropyl cellulose, polacrillin potassium, cross-linked polyvinylpyrrolidone, sodium alginate, sodium starch glycollate, partially hydrolysed starch, sodium carboxymethyl starch and starch. Preference is given to croscarmellose sodium and/or cross-linked polyvinylpyrrolidone, more preference is given to croscarmellose sodium.

The amount of the disintegrant contained in the pharmaceutical composition of can be from 0 to 15%, preferably from 5 to 12% by the total weight of the composition.

Binders include, but are not limited to hydroxypropyl cellulose, hypromellose (hydroxypropyl methylcellulose, HPMC), microcrystalline cellulose, acacia, alginic acid, carboxymethylcellulose, ethylcellulose, methylcellulose, hydroxaethylcellulose, ethylhydroxyethylcellulose, polyvinyl alcohol, polyacrylates, carboxymethylcellulose calcium, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, polyvinyl pyrrolidone and pregelatinized starch. Preference is given to a hydrophilic binder which are soluble in the granulation liquid, more preference is given to hypromellose (hydroxypropyl methylcellulose, HPMC) and/or polyvinylpyrrolidone, most preference is given to hypromellose.

The amount of the binder contained in the pharmaceutical composition of can be from 0 to 15%, preferably from 0.5 to 8% by the total weight of the composition.

Lubricants include, but are not limited to calcium stearate, magnesium stearate, mineral oil, stearic acid, fumaric acid, sodium stearylfumarate, zinc stearate and polyethyleneglycol. Preference is given to magnesium stearate.

The amount of the lubricant contained in the pharmaceutical composition of can be from 0 to 2%, preferably from 0.2 to 0.8% by the total weight of the composition.

Fillers include, but are not limited to dibasic calcium phosphate, kaolin, lactose, mannitol, micro-crystalline cellulose, silicated microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, mannitol, maltitol, sorbitol, xylitol, lactose for example the anhydrous form or the hydrate form such as the monohydrate form, dextrose, maltose, saccharose, glucose, fructose or maltodextrine, powdered cellulose, precipitated calcium carbonate, sodium carbonate, sodium phosphate and starch. Preference is given to microcrystalline cellulose, mannitol, lactose and/or dicalcium phosphate, more preference is given to microcrystalline cellulose.

The amount of the filler contained in the pharmaceutical composition of can be from 0 to 60%, preferably from 3 to 20 % by the total weight of the composition.

Surfactants and Wetting agents include, but are not limited to heptadecaethylene oxycetanol, lecithins, sorbitol monooleate, polyoxyethylene sorbitol monooleate, polyoxyethylene stearate, polyoxyethylen sorbitan monolaurate, benzalkonium chloride, nonoxynol 10, oxtoxynol 9, polysorbates for example 20, 40, 60 or 80, sorbitan mono-palmitate, sodium salts of fatty alcohol-sulfates such as sodium lauryl sulfate, sodium dodecylsulfate, sodium salts of sulfosuccinates such as sodium dioctylsulfosuccinate, partially esters of fatty acids with alcohols such as glycerine monostearate, partially esters of fatty acids with sorbitans such as sorbitan monolaurate, partially esters of fatty acids with polyhydroxyethylene sorbitans such as polyethyleneglycol sorbitan monolaurate, -monostearate or -monooleate, ethers of fatty alcohols with polyhydroxyethylene, esters of fatty acids with polyhydroxyethylene, copolymers of ethylenoxide and propylenoxide (Pluronic®) and ethoxylated triglycerides. Preference is given to sodium lauryl sulfate.

The amount of the surfactant contained in the pharmaceutical composition of can be from 0 to 5 %, preferably from 0.1 to 2 % by the total weight of the composition.

Film-forming agents and coating materials include, but are not limited to liquid glucose, hydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose (hypromellose, HPMC), methylcellulose, ethylcellulose, cellulose acetate phthalate, shellac, polyvinylpyrrolidone, copolymers of vinylpyrrolidone and vinylacetate such as Kollidon® VA64 BASF, copolymers of acrylic- and/or methacrylic acid esters with trimethylammoniummethylacrylate, copolymers of dimethylaminomethacrylic acid and neutral methacrylic acid esters, polymers of methacrylic acid or methacrylic acid esters, copolymers of acrylic acid ethylester and methacrylic acid methyl ester, and copolymers of acrylic acid and acrylic acid methylester. Preference is given to hydroxypropyl methylcellulose (hypromellose, HPMC) as film-forming agent.

Plasticizers include, but are not limited to polyethylene glycol, diethyl phthalate and glycerol. Preference is given to polyethylene glycol.

Further commonly used pharmaceutical exipients which can be used as appropriate to formulate the composition for its intended route of administration include, but is not limited to: Acidifying agents for example acetic acid, citric acid, fumaric acid, hydrochloric acid and nitric acid; alkalizing agents for example ammonia solution, ammonium carbonate, diethanolamine, mono-ethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine and trolamine; adsorbents for example powdered cellulose and activated charcoal; stabilizers and antioxidants for example ascorbic acid, ascorbyl palmitate, butylated hydroxy-anisole, butylated hydroxytoluene, hypophosphorus acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate and sodium metabisulfite; other binding materials for example block polymers, natural and synthetic rubber, polyacrylates, polyurethanes, silicones, polysiloxanes and styrene-butadiene copolymers; buffering agents for examples potassium metaphosphate, dipotassium phosphate, sodium acetate, sodium citrate anhydrous and sodium citrate hydrates; encapsulating agents for example gelatin, starch and cellulose derivates); flavorants, masking agents and odors for example anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin; humectants for example glycerol, propylene glycol and sorbitol; sweeteners for example aspartame, dextrose, glycerol, mannitol, propylene glycol, saccharin sodium, sorbitol and sucrose; anti-adherents for example magnesium stearate and talc; direct compression excipients for example dibasic calcium phosphate, lactose and microcrystalline cellulose; tablet polishing agents for example carnauba wax and white wax.

Preference is given to a pharmaceutical composition comprising the compound of the cyclic dipeptide in a portion of at least 40%, a filler in a portion of from 0 to 60%, a disintegrant in a portion of from 0 to 15%, a binder in a portion of from 0 to 15%, a lubricant in a portion of from 0 to 2% and a surfactant in a portion of from 0 to 5% by weight of the composition.

Fig. 1. LBP-K10 CM inhibited the progress of in situ breast cancer progression in mouse model. 20% LBP-K10 CM diluted with drinking water was supplied as daily drinking water for the first day of transplantation of MDA-MB-231 cells. As early as on the 17_th day, the volume of tumor was obviously different. On the 31_st day, the volume of tumor showed a significantly difference between the 20% LBP-K10 CM supplied group and drink water supplied control group. *P<0.05.

Fig. 2. LBP-K10 CM induced apoptosis in breast cancer MDA-MB-231 cells.

Serial concentration of LBP-K10 CM was employed to treat MDA-MB-231 breast cancer line. Hoechst 33342 (blue) and PI (red) staining were performed for identifying the death of cancer cells. Majority of cell dead with the treatment of 5% LBP-K10 CM. (A) MTT assay was performed to verify the cell viability in the present of different concentration of LBP-K10 CM. *P<0.05. (B) After three days of treatment of 3% LBP-K10 CM, 63.8% of cells arrested in G2/S phase. *P<0.05. (C) The expression of apoptosis pathway related proteins were examined by western blot. The increased expression of cytosolic cytochrome C and BAD and the decreased expression of BCL-2 indicated LBP-K10 CM induced the activation of apoptosis pathway in cancer cells.

Fig. 3. MC-K10 induced caspase-3 dependent apoptosis in breast cancer cells. (A) Serial dilutions of MC-K10 with PBS were employed to treat cancer cells. MTT assay was performed to examine the viability of cells. The trend line also was also shown along with the column diagram. More than half of the cells can survive when the concentration of MC-K10 was less than 20%. (B) Western blot showed caspase-3 elevated along with the increasing expression of cytosolic cytochrome c. (C) The cell viability reversed when caspase-3 inhibitor, Ac-DEVD-CHO was supplied in 20% MC-K10 treatment group. *P<0.05.

Fig. 4. Cyclic dipeptides impaired cancer stem cell activity. (A) 10 mM cyclo(Phe-Pro) or 20% CDPs was employed to treat MDA-MB-231 cells and the sphere formation ability was examined. Both the single cyclo(Phe-Pro) and MC-K10 were capable of impairing sphere formation, though MC-K10 showed a significant higher inhibition activity. *P<0.05. (B) CD133⁺ cell decreased dramatically in MC-K10 treated group, and due to few cells could obtain in passage-3, FACs was not possible performed in MC-K10 treated group. However, the CD133⁺ cell partition in passage-2 and passage-3 was comparable in cyclo(Phe-Pro) treated group. *P<0.05. (C) Passage-2 sphere was lysed to examine the expression of Oct4. (D) The western blot results of (C) were quantified. MC-K10 treatment significantly diminished the expression of Oct4 than cyclo(Phe-Pro) treatment, though cyclo(Phe-Pro) also can induce a dramatic decreasing expression (38.4%) of Oct4 when comparing with intact control group. *P<0.05.

Material and methods

2.1. Culturing and identifying lactic acid bacteria, MC-K10 and cyclo(Phe-Pro) collection

Lb. plantarum LBP-K10 were isolated, cultivated and identified as described before. Organic acids was removed through the weak anion exchanger Amberlite IRA-67 as previously proposed. The resulting eluents from the culture supernatant were lyophilized and were extracted with 5-fold volumes of methylene chloride. After the methylene chloride-extracted fractions were evaporated, the resulting powder was dissolved in the appropriate amount of sterilized distilled water and then diluted with PBS to the original volume of culture filtrate employed in the very beginning. This solution was regarded as 1 x starting solution and designated as MC-K10 for further experimental trials.

2.2. Cancer cell and Animal studies

Human breast cancer cell MDA-MB-231 (ATCC, Manassas, VA) were cultivated as described. SCID mice were used as 6 weeks of age and 10 mice for each group. All mouse studies were performed in accordance with protocols approved by the Animal Care and Use Committee at Southeast University (Approval ID: SEU-20150615-3). 2 x 10⁶ MDA-MB-231 cells were injected into mammary fat pad in a 100 μL volume of sterile phosphate-buffered saline. LBP-K10 CM were diluted 5 times with drinking water and given to the mice on the first day of cell transplantation. Tumors were measured every two weeks with precision calipers. Tumor volume was calculated at: volume =(length x width²)/2.

2.3. Flow cytometry

For flow cytometry, cells were treated with MC-K10 or CDP for 3 days. After 3 washes with PBS, cells were detached for 10 min at 37 degree Celsius with trypsin (Gibco). Cells were stained with fluorescent antibody CD133 (Becton-Dickinson, San Jose, CA, USA) and analyses were performed with FACSCalibur (Becton-Dickinson, NY, USA).

2.4. Cell cycle analysis

2 x 10⁶ cells were fixed in cold methanol and stained with presidium iodide (PI, Sigma). Cells were analyzed for DNA content by EPICS-XL scan (Beckman Coulter) by using doublet discrimination gating. All analyses were performed in triplicate and 20,000 gated events/sample were counted.

2.5. Cell viability assay

Hoechst 33342 (Sigma-Aldrich, St. Louis) and PI were used for staining cells treated in the mentioned condition following the method described before with slight modifications [44]. Cells were fixed in 1% glutaraldehyde for 30 min at room temperature. The cells were then stained with 5 μg/ml Hoechst 33342 in PBS for 30 min at room temperature. After further incubated with 1 μl PI, the cells were immediately observed using a fluorescence microscope.

For MTT assay, the indicated cells were incubated in MTT (50 μl of 2 mg/ml) in PBS for 4 h at 37 degree Celsius. DMSO (120 μl, Sigma) was added to each well and incubated 1 h on an orbital shaker. The result was read at 570 nm with an Ultra-microplate reader (ELx 808; Bio-Tek Instruments, Winooski, VT).

2.6. Western blot analysis

After extracted from the cells, the protein concentrations were measured and10 μg of protein extracted was loaded on 10% SDS-PAGE. The separated proteins were transblotted onto a polyvinylidene fluoride (PVDF) membrane. The membrane were blocked in 5% nonfat milk with 0.05% Tween-20. Then, the blot was incubated as follws: all of the antibodies were purchased from Cell Signaling; a polyclonal rabbit anti-human Bcl-2-associated death promoter (BAD) antibody, a rabbit anti- human caspase-3 antibody, a rabbit anti- human Bcl-2 antibody, a rabbit antihuman Cytochrome C antibody, a rabbit anti-human CD133 antibody, a rabbit anti-human Oct4/3 antibody and a rabbit anti-human beta- actin antibody at 1≥1000 dilutions in 5% blocking solution over night at 4 degree Celsius. An anti-rabbit IgG antibody was used as a secondary antibody at a dilution of 1≥5000. The results were detected with an enhanced chemiluminescence kit (ECL; Amersham Bioscience/GE Healthcare, Little Chalfont, UK).

2.7. Sphere forming assays

Single-cell suspensions were obtained from cell monolayers and counted with hemocytometer. Single-cell suspensions (10⁵ cells per 100 mm dish) were plated with an ultralow attachment surface. Triplicate experiments were performed. Wells containing single-cell suspension were chosen for cultivation. Nine days later, Colonies of at least 60 μm in diameter (determined by using an eyepiece graticule with crossed scales) were counted.

2.8. Statistical analysis

Data are presented as means ± standard error (SD) in quantitative experiments. The differences between groups were analyzed using the unpaired Student's t-test. P values <0.05 were considered significant.

3. Results

3.1. Breast cancer prevention effect of LBP-K10 CM in mouse model

Lactic acid bacteria was isolated and cultured following the method described previously [36]. The pH of culture was dramatically decreased dramatically from pH7 to 4.0 along with the cell number expansion. After 24 h the cell reached their stationary station and the pH value was around 3.8-4.0. The medium was collected and heat inactivated for after 72 h further studies. The expanded bacteria was characterized by gene sequencing and was confirmed as Lb. plantarum LBP-K10 as mentioned in our previously studies [36,37].

After centrifuging, cells were removed from the heat inactivated Lb. plantarum LBP-K10 cultures. This cell- free Lb. plantarum LBP-K10 conditioned medium was diluted 5 times with drinking water. This 20% LBP-K10 CM water was supplied to the mice on the first day of cancer cell transplantation. On the day of 31_st, the volume of the tumors in LBP-K10 CM supplied group was significantly smaller than that in the positive control group. The obvious cancer inhibition effect could be observed on the 10_th day after cell transplantation. This inhibition effect become significant on the day 17, meanwhile the tumor growth rate increased in the control group during the same time period. However, though LBP-K10 CM water was continuously supplied till the 31_st day, the tumor sizes were maintained since the 17_th day, while tumors keep growing in the positive control group (Fig. 1).

3.2. Cell-free LBP-K10 CM induced cell death on breast cancer cell line MDA-MB-231

The in vivo studies encouraged us to explore the mechanism of breast cancer prevention effect of the LBP-K10 CM. First, we treated the triple negative breast cancer cell line MDA-MB-231 with various concentration of LBP-K10 CM. Majority of cells were lysed in vitro when cultured in conditions with more than 5% LBP-K10 CM supplement (Fig. 2A). The viability of cancer cells was quantified with MTT assay. Around 80% of cancer cells could survive after 3-day treatment with 3% LBP-K10 CM, whereas only 30% of cells could survive in 5% treatment (Fig. 2B). Further studies were executed in 3% LBP-K10 CM. The cell cycle states were checked with FACs (Fig. 2C). The expressions of proteins that are evolved in apoptosis pathway, BAD, BCL-2 and cytosolic cytochrome C were examined with western blot. The results showed the expression of cytosolic cytochrome C and BAD were increased while BCL-2 expression level was decreased (Fig. 2D). The results revealed that cytosolic cytochrome C induced apoptosis might be active after 3% LBP-K10 CM treatment.

3.3. MC-K10 from Lb. plantarum LBP-K10 effectively induced breast cancer cell MAD-MB-231 apoptosis

Our previous studies have displayed Lb. plantarum LBP-K10 isolated from fomented food produce several kinds of CDPs. Single CDP components or MC-K10 conditioned by LAB has been proved to have antibiotic effect. Since studies in other group also proved that CDPs are capable of inducing cancer cell apoptosis, we wondered whether the MC-K10 derived from Lb. plantarum LBP-K10 possessed anticancer effect.

The CDPs enriched fraction, MC-K10, was diluted to the original volume of LBP-K10 CM with PBS and regard as 1 X solution. This solution was diluted with PBS as mentioned in further studies. Serial dilutions were tested on cancer cells. As shown in MTT assay, 57% cancer cell can survive in 20% MC-K10 supplement conditions, however, only around 10% cancer cell can survive in 40% MC-K10 supplement conditions (Fig. 3A). We further tested the expression of apoptosis related proteins, capase-3 and cytosolic cytochrome C. The expression of both proteins were elevated when the cells treated with 20% MC-K10 (Fig. 3B). To further confirm caspase-3 pathway involved in MC-K10 induced apoptosis, Ac-DEVD-CHO, an inhibitor of caspase-3, were employed in MC-K10 treatment group. As a result, the survival rate was reversed to 82% (Fig. 3C). These results indicate MC-K10 isolated from LBP-K10 CM could induce breast cancer cell apoptosis through caspase-3 pathway.

3.4. Cyclic dipeptides produced by LAB impaired cancer stem cells viability and tumor formation ability

Cancers are initiated and maintained by cancer stem cells. Substances that can effectively kill the cancer cells may be resisted by cancer stem cells. In vivo and in vitro studies mentioned above encouraged us to test the effect of cyclic dipeptides on cancer stem cells. Cyclo(Phe-Pro), which is the most abundant component among CDPs in the culture filtrates of LBP-K10, was purified in this study. Serial sphere formation assays were performed. In the first passage of sphere formation test, the sphere formation ability was 43% in cyclo(Phe-Pro) treated group when comparing with control group. However, the sphere formation ability in MC-K10 was significantly decrease to 45%. In cyclo(Phe-Pro) treated group, there is a significant difference in sphere formation ability between passage-1 and passage-2, whereas this sphere formation ability was comparable between passage-2 and passage-3. The sphere formation of MC-K10 treated group was continuously decreased in passage-2 and passage-3 (Fig. 4A). Since CD133 was regarded as cancer stem cell marker, the partition of CD133⁺ cells were analyzed. CD133⁺ cells were decrease dramatically in MC-K10 treated group (Fig. 4B). We next examined another cancer stem cell marker, Oct4 and quantified the western blot results. Oct4 expression were decreased in both cyclo(Phe-Pro) and MC-K10 treated group. Comparing with cyclo(Phe-Pro) treatment, the expression of Oct4 was dramatically down regulated (Fig. 4C and D). These results indicated MC-K10 might impair the stemness and self-renew ability of breast cancer stem cells.

4. Discussion

LAB can survive in acid environmental and resist to the digest effect of bile, thus, it can pass through the GI track and adhere on the intestine cells. Since colon cancer is one of the major types of GI track cancer and LAB may directly interact with colon cancer in GI tack, studies focus on the anti-colon cancer effect of live LAB. Several mechanisms have been revealed. Very few studies investigated the effect of LAB on breast cancer. Previously, we confirmed a special set of cyclic dipeptide complex producing by Lb. plantarum LBP-K10, which give a strong effect of anti-microorganism. Despite the anti-microorganism effect, other studies confirmed cyclic dipeptides were capable of inducing cancer cell apoptosis. Most importantly, studies revealed the quickly absorption of cyclic dipeptides in animal. Therefore, we purposed that oral administration LBP-K10 CM might inhibit the growth of breast cancer. This study proved that oral administration LBP-K10 CM may effectively arrest the growth of breast cancer cell growth in animal model.

LBP-K10 CM contained a large amount of organic acid that contribute to cancer prevention effect of Lb. plantarum LBP-K10, such as butyrate acid. Since the anticancer effect of organic acid has been well elaborated, we further tested the anticancer effect of MC-K10 secreted by Lb. plantarum LBP-K10. As our expectation, MC-K10 effectively inhibited the growth of breast cancer cell.

Previously, we tested the effect of sodium butyrate, which is one of compound of organic acid complex produced by Lb. plantarum LBP-K10, on breast cancer prevention. In this study, we interested on the effect of MC-K10 on cancer stem cells. Our results indicated MC-K10 treatment or cyclo(Phe-Pro) may induce the apoptosis of breast cancer stem cells. The in vivo results displayed that Lb. plantarum LBP-K10 administration could not eradiate breast cancer. This may give a clue that cancer stem cells are heterogeneous. Further studies need to be done for discovering the mechanism beneath these results.

Above of all, we proved that orally applying LBP-K10 CM was helpful for inhibiting the growth of in situ breast cancer in mouse model and CDPs participated in this inhibition effect. MC-K10 conditioned Lb. plantarum LBP-K10 and cyclo(Phe-Pro) were capable of inducing apoptosis of cancer cells. Our study displayed the inhibition effect of LAB on non-GI Track cancer by oral administration and CDPs administration produced by Lb. plantarum LBP-K10 was promising route for cancer therapy due to their inhibition effect targeting cancer cells.

This study indicated that oral supplying LAB may contribute for breast cancer prevention and CDP molecules might have the potential for cancer therapy by targeting cancer stem cells. This could contribute for developing new drugs depending on CDP structure for cancer prevention.

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Claims (7)

1. Composition for prevention of breast cancer comprising cyclic dipeptide.
2. The composition for prevention of breast cancer according to claim 1, wherein the cyclic dipeptide cyclo(Phe-Pro).
3. The composition for prevention of breast cancer according to claim 1, wherein the cyclic dipeptide is produced from Lb. plantarum LBP-K10.
4. The composition for prevention of breast cancer according to claim 3, wherein Lb. plantarum LBP-K10 is sterilized after the production of cyclic dipeptide..
5. The composition for prevention of breast cancer according to claim 3, wherein the composition for prevention of breast cancer is prepared comprising the steps:

   producing cyclic dipeptide with Lb. plantarum LBP-K10 in culture medium;

   sterilizing the said Lb. plantarum LBP-K10 by heat treatment; and

   removing the sterilized Lb. plantarum LBP-K10 from the culture medium by centrifugation.
6. A method of preparation of composition for prevention of breast cancer comprising the steps of;

   culturing Lactobacillus sp.in a culture medium;

   sterilizing the Lactobacillus by heat treatment; and

   removing the sterilized Lactobacillus sp. from the culture medium.
7. The method of preparation of composition for prevention of breast cancer according to claim 6, wherein the method further comprise the step of concentrating the cyclic dipeptide.

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